Classical swine fever (CSF), previously referred to hog cholera (HC), is an important infectious disease of swine caused by CSFV. Domestic pigs and wild boar are susceptible to CSFV. The virus belongs to the genus Pestivirus within the family Flaviviridae, which also includes bovine viral diarrhea virus (BVDV) and border disease virus (BDV) (Matthaeus, Zbl. Vet. Med. 328: 126-132, 1981). They are antigenically and structurally closely related but CSFV can be distinguished from BVDV and BDV in serological and nucleotide differences (Paton, J Comp pathol 112: 215-236, 1995). The most commonly used in laboratory CSF diagnostic methods, such as immuno-fluorescence stain, enzyme linked immunosorbent assay (ELISA) and RT-PCR, can hardly distinguish the field isolates of CSFV from the lapinized CSF vaccine viruses. Records of CSF in Taiwan date back to 1938 (Lee, Scientific Agri (Taiwan), 2(11): 4-14). In an effort to control this highly contagious disease, a live attenuated vaccines made from a lapinized CSF vaccine virus, LPC strain, have been widely used in the field since 1958 in Taiwan (Lin, National Science Council Special Publication Number 5, 1-42, 1981). Vaccination significantly decreased the incidence. However, sporadic outbreaks were still reported occasionally. According to the current legislation on CSF in Taiwan, each piglet has to be vaccinated twice with the LPC vaccine virus in 3, 6 or 6, 9 weeks old, it depend on the decrease level of maternal antibody. Unfortunately, the vaccine virus can be detected and can't be differentiated from field isolates of CSFV by ELISA and RT-PCR in samples of pigs. Since the LPC vaccine virus interfered with the laboratory diagnosis of CSF, the RT-PCR amplicons always proceed with nucleotide sequencing to exclude the interference by LPC vaccine virus in Taiwan.
A tentative assignment of world isolates of CSFV by genotyping has been divided it into three groups with three or four subgroups: 1.1, 1.2, 1.3; 2.1, 2.2, 2.3; 3.1, 3.2, 3.3, 3.4 (Paton, Vet. Microbiol. 73: 137-157, 2000). Phylogenetic analysis of the Erns and E2 sequences of 158 CSFVs, which were isolated in the field in Taiwan between 1989 and 2003, shows that four distinct CSFVs genotypes existed in Taiwan including one endemic strain (subgroup 3.4) and three introduced strains (subgroup 2.1a, 2.1b and 2.2). The analysis also shows the LPC strain doesn't belong to the aforementioned four subgroups, but to the subgroup 1.1. (Pan, Arch Virol, 150(6): 1101-19, 2005).
There are four lapinized CSF vaccine viruses, namely LPC, HCLV, Chinese C and Riem C, widely utilized in the world nowadays. The LPC strain was derived from the Rova strain of CSFV, which had already undergone about 250 serial passages in rabbits by Lederle Laboratory in Philippines and was introduced into Taiwan by Dr. Chung-Tao LEE in 1952 (Lee, Scientific Agri (Taiwan), 2(11): 4-14). The pigs inoculated with this virus showed a severe post-vaccinated reaction and a few of them even died of CSF after vaccination. In order to obtain a highly safe and potent strain for CSF vaccination, the virus was then rapidly and carefully serial-passaged through native Taiwan rabbits. After more than 800 passages in rabbits, it proved extremely safe for pigs and highly effective against CSFV (Lin, National Science Council Special Publication Number 5, 1-42, 1981). Nowadays, the LPC vaccine is widely utilized to protect pigs from CSF in Taiwan. The HCLV strain was derived from the wildtype strain Shimen by 480 passages in the bodies of rabbits in China in 1950s. (Wu, Virus Genes, 23(1): 69-76, 2001). The Chinese C strain is a cell culture adapted derivative of HCLV strain. (Oleksiewicz, Veterinary microbiology, 92:311-325, 2003). The Riem C strain is a cell culture adapted derivative of HCLV strain and used as bait vaccine in Europe (Oleksiewicz, Veterinary microbiology; 92:311-325, 2003). Wu et al. (Virus Genes, 23(1): 69-76, 2001) have sequenced HCLV strain and discovered one notable insertion of 12 continuous nucleotides, CTTTTTTCTTTT (SEQ ID NO:8) in the 3′-untranslated region of HCLV genomic cDNA when compared with its parental virulent Shimen strain. Wong et al (Virus Genes; 23(2): 187-92, 2001) also sequenced the whole genome of LPC vaccine strain and found that an insertion 0113 nucleotides, TTT(C/T)CTTTTTTTT SEQ ID NO:9, in the 3-untranslated region of LPC vaccine strain. The inventors of the present invention had also compared all the CSFV sequences from the GenBank and found that only the four lapinized CSF vaccine viruses, LPC, HCLV, Chinese C and Riem C strains, have an insertion of 12˜13 nucleotides in their 3-untranslated regions and the insertion is not found in the field isolates of CSFV. Other non-lapinized CSF vaccine viruses, such as Japanese GPE- and Russian CS vaccine strains, also do not have the insertion.
Vaccination is one of the most successful tools for the prevention of CSFV infection. Unfortunately, the use of laboratory diagnostic methods to detect CSFV could be interfered by vaccine virus when attenuated vaccines are in use. For this reason it is of interest to know how long after vaccination can the vaccine strain be detected in samples that are commonly used for diagnostic procedures. To study the duration of vaccine virus distribution in piglets, Lorena et al. (Veterinary microbiology, 81: 1-8, 2001) inoculated piglets with the Chinese C strain vaccine virus and studied the distribution of vaccine virus in organ samples of inoculated piglets. He found that the virus can be detected in tonsil on post-inoculation day (PID) 6, 8, 10, 13 and 16 using ELISA and in blood samples on PID 2, 4, 6, 8, 10, 13, and 16 using RT-PCR. Therefore, he emphasized that this factor must be considered in routine diagnostic procedure, when vaccination against CSF with a live vaccine is carried out. In Germany, CSF was present in wild boar in different federal states (Veterinary microbiology, 82: 301-310, 2001). Infection in domestic pigs was usually caused by direct or indirect contacts with infected wild boars. Wild boars distributed in the woods and they are difficult to be caught for injecting CSF vaccine. Therefore, Oral application of CSF vaccines (lapinised or cell culture vaccines) is necessary and has been investigated in Europe (Veterinary microbiology, 73: 239-252, 2000). Kaden et al. (J Vet Med B Infect Dis Vet Public Health, 51(6): 260-2, 2004) studied the persistence period of the Chinese C strain vaccine virus in immunized animals after oral vaccination. The results show that the C strain virus can be found in organs until day 8 post-vaccination (pv) in domestic pigs and until day 9 pv in wild boars. In the CSF endemic countries like Taiwan where vaccination program with live vaccine is carried out, the vaccine virus can probably be detected in the blood and lymphatic tissue samples such as tonsil, lymph nodes and spleen. Therefore, diagnosis with the commonly used diagnostic methods such as immuno-fluorescence stain, ELISA and RT-PCR can be interfered by the vaccine virus.
Virus isolation, ELISA and RT-PCR are the most commonly used methods for CSF laboratory diagnosis. Paton et al. (Veterinary microbiology 73: 159-174, 2000) show that the order of the sensitivity was RT-nested PCR>RT-PCR>virus isolation>ELISA when applying these methods to clinical samples in CSF diagnosis. Dewulf et al. (Journal of Virological Methods 119: 137-143, 2004) compare several CSF laboratory diagnostic techniques on live animals for detection of infection. He concluded that the RT-nPCR technique is the best diagnostic tool available for early detection of a CSF infection. A real-time RT-PCR for the simple and rapid diagnosis of CSF has been developed and evaluated in experimentally infected swine and clinical samples (Risatti, Journal of Clinical Microbiology; 41(1): 500-505, 2003; Risatti, Journal of Clinical Microbiology, 43(1): 468-471, 2005). Accordingly, real-time RT-PCR is recognized as a sensitive method for rapid diagnosis of CSF. However, no real-time RT-PCR for distinguishing the field isolates of CSFV from the lapinized CSF vaccine viruses has been established.
The RT-PCR and nucleotide sequencing are widely used as the methods to solve this problem. The disadvantage of these methods includes the laborious process of the methods and the incapability of screening large field samples. Zaberezhny et al. (Dtsch Tierarztl Wochenschr. September; 106(9): 394-7, 1999) have used RT-PCR and restriction enzyme digestion for differentiation between Russian vaccine strain from field isolates of CSFV. Vilcek et al. (Acta Vet Scand. 39(3): 395-400, 1998) also used restriction endonuclease cleavage of PCR amplicons to distinguish the vaccine strain from European field strains. These are the two documents available about the utilization of the RT-PCR and restriction enzyme digestion method to differentiate the vaccine virus and field isolates of CSFV; besides, the prior art can only distinguish the two viruses by RT-PCR followed by nucleotide sequencing. Thus there is no prior disclosed information concerning the utilization of the characteristic of one 12˜13 nucleotides inserting in the genome of the 3′-untranslated region of the lapinized CSF vaccine viruses to establish a differential RT-PCR without combination with other technique. CSFV specific primers are designed to amplify the aforementioned 3′-untranslated region and then the size of the RT-PCR amplicons can be compared directly by electrophoresis without further processing the complicated enzymatic digestion and nucleotide sequencing to determine the existence of the field isolates of CSFV and lapinized CSF vaccine viruses and to differentiate between them. The diagnosis of CSF can thus be more rapid, convenient and the interference from the vaccine virus can be more correctly excluded.